key: cord-0980328-7rzvss6r
authors: Lorenzo, Ramiro; Defelipe, Lucas A.; Aliperti, Lucio; Niebling, Stephan; Custódio, Tânia F.; Löw, Christian; Schwarz, Jennifer J.; Remans, Kim; Craig, Patricio O.; Otero, Lisandro H.; Klinke, Sebastián; García-Alai, María; Sánchez, Ignacio E.; Alonso, Leonardo G.
title: Deamidation drives molecular aging of the SARS-CoV-2 spike receptor-binding motif
date: 2021-05-21
journal: bioRxiv
DOI: 10.1101/2021.05.20.445042
sha: 8fd4c809dacfb17874396ea0cb242e5cb2d20dac
doc_id: 980328
cord_uid: 7rzvss6r

The spike is the main protein component of the SARS-CoV-2 virion surface. The spike receptor binding motif mediates recognition of the hACE2 receptor, a critical infection step, and is the preferential target for spike-neutralizing antibodies. Post-translational modifications of the spike receptor binding motif can modulate viral infectivity and immune response. We studied the spike protein in search for asparagine deamidation, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, affecting both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike. Similar deamidation hotspots are frequently found at the spike receptor-binding motifs of related sarbecoviruses, at positions that are mutated in emerging variants and in escape mutants from neutralizing antibodies. Asparagine residues 481 and 501 from the receptor-binding motif deamidate with a half-time of 16.5 and 123 days at 37 °C, respectively. This process is significantly slowed down at 4 °C, pointing at a strong dependence of spike molecular aging on the environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the hACE2 receptor more than 3.5-fold. A model for deamidation of the full SARS-CoV-2 virion illustrates that deamidation of the spike receptor-binding motif leads to the accumulation in the virion surface of a chemically diverse spike population in a timescale of days. Our findings provide a mechanism for molecular aging of the spike, with significant consequences for understanding virus infectivity and vaccine development.

In December 2019, a viral pneumonia outbreak was reported in Wuhan, China (1) . This outbreak quickly turned into a pandemic disease (COVID-19) of international concerns (2) , and the novel pathogen causative of a Severe Acute Respiratory Syndrome (SARS) was soon identified as SARS-CoV-2, a new member of the Betacoronaviruses genera. SARS-CoV-2 and SARS-CoV, the agent responsible for the 2002-2003 pneumonia outbreak, are closely related to the bat coronaviruses from which they likely originated and passed to an intermediate species that ultimately infected humans (3, 4) . The host specificity and infectivity of SARS-CoV-2 and SARS-CoV rely on the spike protein (S).Through its receptor-binding domain (RBD, residues 319 to ~515), S recognizes the human Angiotensin-converting enzyme 2 (hACE2) with nanomolar affinity, triggering events that culminate with the fusion of the cellular and viral membranes (5) . In SARS-CoV-2, the S protein is synthesized as a 1273-residue heavily glycosylated polypeptide that is cleaved by the host furin protease between the S1 (1-685) and S2 (686-1273) subunits (6) . On the surface of native viruses, the S protein is mainly observed as a metastable trimer in the prefusion conformation. The RBD of each S protomer can switch between a receptoraccessible conformation known as the "up-state"and a receptor-inaccessible and buried conformation that packs against the N-terminal domain (NTD) of the neighboring protomer called the "down-state" (7) . Two regions can be identified in the RBD, a conserved core and a more variable region termed as the receptorbinding motif (RBM, residues 438-506). The latter region contains residues that establish direct contact with hACE2, determining S protein affinity and specificity (8) .

Inter-species spillover is often observed in the coronavirus family members, a phenomenon that mainly originates from amino acid mutations in the RBD that enables S to bind ACE proteins from two different host species (9, 10) . Beyond S crucial role in restricting viral host infectivity, the protein is the target of potent neutralizing antibodies (11) (12) (13) with therapeutic use and the main antigenic component of vaccines (14, 15) . It is of particular interest to understand how mutations and posttranslational modifications (PTM) in RBD affect viral infectivity, generate antigenic escape variants or restrict the humoral and cellular immunity.

Asparagine (Asn) deamidation is a spontaneous and irreversible frequently observed PTM (16, 17) . As a result of the substitution in the Asn side-chain of the carboxamide nitrogen atom by a hydroxyl group, a mixture of aspartic and isoaspartic acid (a beta amino acid) is generated (18) , introducing a negative charge and a rearrangement of the protein backbone in the latter case. The deamidation rate, which heavily depends on the primary sequence and local structure, can be estimated using bioinformatic tools that rely on different approaches such as structural constraints, machine-learning or primary sequence and disorder predictors (19) (20) (21) . The fastest deamidation rate is often observed for the asparagine-glycine (NG) dipeptide in loosely structured regions, which are generally regarded as deamidation "hotspots" for which most of the methods provide accurate prediction (22) .

The S protein contains multiple deamidation sites, but only some of them bear biological relevance. To identify these relevant sites, we applied a prioritization process based on four key points: (i) In-silico identification of hotspots and deamidation rates using the NGOME-LITE algorithm (23), (ii) conservation of the sites among Betacoronaviruses, (ii) location of the sites at the ACE2 binding surface, and (iv) experimental determination of the deamidation rates at specific hotspots by mass spectrometry.

A direct consequence of deamidation events in Asn residues located at the RBM is the modification of the electrostatic potential of the receptor-binding surface due to the introduction of a negative charge from the aspartic or isoaspartic carboxylate. Consequently, as the RBD is the preferential region from the S protein for neutralizing antibodies (11) (12) (13) , deamidation could also affect the efficiency of the humoral immune response. With these data we generated a model that computes the proportion of deamidated protomers in a single viral particle and predicts how these species evolve. Our findings shed light on deamidation, an aging mechanism that operates on the RBM, a critical region that determines virus infectivity and may have a profound impact on vaccine development.

We initially estimate the individual deamidation (Fig. 1A) half-time (t1/2) using the NGOME-LITE (23) algorithm, for all asparagine residues with no glycosylation probability. We further include positional information and the conservation degree of deamidation-prone Asn to generate a deamidation profile for a set of Betacoronavirus S proteins (Fig. 1B) . We restricted our analysis to a group of S proteins from Betacoronavirsuses with demonstrated affinity for the hACE2 receptor, which includes SARS-CoV-2 (Wuhan), SARS-CoV (Urbani), SARS-CoV (GZ0402), the closely related Bat SARS-like CoVs RaTG13 (24) and WIV1 (25) . SI Appendix, Table S1 (Genbank accession number, references and conservation).

A similar deamidation profileis observed for the five S proteins (Fig. 1B) , characterized by a major group of Asn residues in a slow-deamidation regime with estimated t1/2 greater than 100 days and a restricted group of residues in a fast-deamidation regime, with estimated t1/2 ranging between 20 to 35 days ( Fig. 1B and SI Appendix, Table S2 ). All Asn residues in the fast-deamidation regime (hereafter referred to as deamidation hotspots) are part of NG dipeptides (SI Appendix, Fig. S1 ), for which a deamidation t1/2 of 1.2 days is expected in the absence of structural protection (i.e. in a short non-structured peptide) (18, 21) indicating that the S protein fold protects these Asn residues from deamidation. Slow regime residues virtually do not deamidate during the (expected) viral-life cycle and are devoid of any functional consequence.

Except for the single deamidation hotspot observed at position 74 in the Bat-CoV RaTG13 (in SARS-CoV-2 asparagine residue 74 is glycosylated, (26)), deamidation hotspots are restricted to a group of residues clustered between residues 481 and 501 in the RBM, including residue 493 (only observed in the GZ0402 SARS-CoV), which shows partial conservation among analyzed CoVs, and a group of strictly conserved residues at positions 544, 856 and 907 ( Fig. 1C and SI Appendix, Fig. S1 ), observed in all the selected S sequences. Hotspots within the RBM are, altogether, termed as the RBM deamidation cluster. Overall, the deamidation profile shows that hotspots are not evenly distributed along these S protein sequences ( Fig.   1B and Fig. 1C ).

Beyond sequence context, the protein fold, backbone flexibility and surface accessibility correlate with the experimental Asn deamidation rates (27, 28) . The hydrolysis of the cyclic intermediate is required to complete the deamidation reaction (Fig. 1A) for which solvent molecules must reach the reaction center.

Solvent accessibility can be assessed by calculating the Relative Accessible Surface Area (RASA) of the deamidation-prone residue to a small probe radius of 1.4 Å. In addition, NGOME-LITE is unable to sense the effect of the tertiary and quaternary structure on the deamidation rate, factors that become of critical importance for hotspots centered at the inter-domain or inter-protomeric interfaces of the S trimer. Higherorder structure factors affecting deamidation rates can be identified by evaluating residue accessibility to a 3.0 Å radius probe.

To discriminate between the predicted deamidation-prone residues that are solvent accessible and those that are present at the inter-domain surface we calculated the side-chain RASA for all deamidation hotspots in the SARS-CoV-2 S structure. We performed our analysis using a structure of the furin-cleaved S protein in the pre-fusion conformation, which contains two RBDs in the down-state and one in the upstate (PDB 6zgg (24) using two probes with radii of 1.4 and 3.0 Å(29) (SI Appendix, Table S3 ).

All five deamidation hotspots are fully or partially accessible to the 1.4 Å radius probe (SI Appendix, Table   S3 ), albeit to a different extent and depending on the RBD conformation, indicating that they are accessible to water molecules, a necessary requirement for hydrolysis. On the other hand, only hotspots Additionally, Asn 907 located in the heavily glycosylated stalk of S is partially accessible to the 1.4 Å radius probe through an internal channel formed by the three protomers, whereas Asn 856 is packed between the contact interface of two protomers (Fig. 1C ). Both positions are fully inaccessible to the 3.0 Å radius probe, irrespective of the RBD conformation.

The deamidation profile of SARS-CoV-2 S obtained with NGOME-LITE shows the presence of conserved deamidation hotspots at relevant protein-protein recognition interfaces. However, the fact that these sequence stretches are part of a large multidomain protein, which is not accounted for by the NGOME-LITE algorithm reduces the prediction accuracy of this bioinformatic tool.

A first experimental observation of the occurrence of deamidation at the predicted RBD hotspots was reported previously by the Wells lab (26) . To monitor N-glycan occupancy in the full-length S protein, they observed 18.9% of 18 O-Asp conversion at Asn 501, 4.8% at Asn 481 and 7.8% at Asn 544 attributable to deamidation, since these hotspots lack canonical glycosylation sequons (N-X-S/T). The amount of 18 O-Asp conversion correlates well with the deamidation t1/2 predicted by NGOME-LITE for all five deamidation hotspot SI Appendix, Table S4 .

As the deamidation kinetics is critical to evaluate the potential effect of the aspartic/isoaspartic formation in the S function, we experimentally assessed the deamidation t1/2 for the hotspots 481, 501 and 544 in a recombinantly expressed extended version of the SARS-CoV-2 RBD construct encompassing residues 319 to 566 (30) at 4 ˚C and 37 ˚C.

It should be noted that residue 544 is not part of the RBD but instead it belongs to the structural conserved subdomain 1 (SD1) that encompasses residues ~516 to ~591 not fully present in our construct. Hence, SD1 may be devoid of its native structure and the interpretation of the observed t1/2 for the 544 hotspot should be treated with caution. In our experimental set-up, the recombinant RBD protein was incubated at pH 7.4 over several days at two different temperatures and the presence of deamidated species was identified and quantified by mass spectrometry (31) (SI Appendix, Fig. S2 and Table S5 ). Fig Deamidation half-times of 16.5±3.7, 123±23, and 7.9±1.2 days were obtained at 37˚C for the 481, 501 and 544 hotspots. Under our experimental conditions, the RBD hotspot 501 remains stable against the deamidation process despite being identified a priori by the NGOME-LITE as the fastest deamidation hotspot. On the contrary, Asn 544 has the highest deamidation rate, with a value close to the expected deamidation rate of a NG sequence in an unstructured peptide model (1.2 days) supporting that this residue is placed in an unstructured region. The disagreement between experimental determined deamidation rates and computational predictions for hotspots that share the common NG sequence highlight that protein structure and associated dynamicsin S can fine tune the deamidation rate.

As expected, the deamidation reaction was slowed down considerably by decreasing the temperature to 4˚C (SI Appendix, Table S4 ). The deamidation half-time for Asn 481 at 37˚C compared to 4 °C is more than 20-fold higher, highlighting that temperature can critically affect the identity of the RBM.

Deamidated species for hotspots 481 and 544 elute at two different retention times (RT) in a reverse phase (RP) chromatography experiment (SI Appendix, Fig. S2 ), in agreement with the reaction mechanism that proposes the generation of a mixture of aspartic or isoaspartic bearing peptides. In RP chromatography, deamidated species containing isoaspartic residues are reported to elute faster than aspartic containing peptides (18) and, based on this chromatographic behavior, the deamidation reaction (20 days at 37 °C) at hotspots 481 and 544 shows an abundance of 46.6% and 68.5% of isoaspartic containing species, respectively (SI Appendix, Table S4 ). On the contrary, only one peak was observed for the deamidated species at Asn 501.

We then evaluated how deamidation affects the affinity of RBD for the ectodomain of hACE2. To this end we performed a bio-layer interferometry (BLI) assay using an aged and heterogeneously deamidated RBD sample with ~65%, ~12% and ~83% of hotspots 481, 501 and 544 in its deamidated form. We observed that the unaged RBD binds hACE2 with an affinity dissociation constant (Kd) of 23.4±0.8 nM whereas the aged RBD has a reduced affinity of only 81.4±0.8 nM (Fig. 2 B) showing that deamidation is detrimental for hACE2 binding and might impact virus infectivity.

Due to the critical role of the RBM residues in determining hACE2 affinity and host-specificity (32, 33) , we further evaluated the conservation pattern of hotspots at the RBM deamidation cluster by extending our analysis to a group of Betacoronaviruses representative of the subgenus Sarbecoviruses (34, 35) which, in addition to SARS-CoV and SARS-CoV-2, includes numerous bat and pangolin viruses ( Fig. 3A and SI Appendix, Table S6 ).

Overall, the RBM deamidation cluster shows a variable conservation pattern ( The overall folds of SARS-CoV and SARS-CoV-2 RBMs are similar with 14 residues (32) observed in equivalent positions in both structures located at less than 5Å from hACE2 binding residues (cut-off for considering a direct contact) including the 493 and 501 deamidation hotspots (Fig. 3B) . Asn 493 and 501 form a direct interaction with hACE2 residues K31 and K353, respectively, which are considered critical for S binding. Mutational studies (32, 33, 36) have shown the relevance of these two positions in determining ACE2 affinity. Furthermore, and of note, the N501Y mutation that eliminates the deamidation hotspot is a hallmark of the emerging SARS-CoV-2 VOC 202012/01 (B.1.1.7) and 501Y.V2 (B.1.351) variants that are rapidly spreading in the UK and South Africa (37) . Both variants are likely to have arisen independently and are associated with an increased transmissibility.

A deamidation hotspot at position 501 is repeatedly observed in SARS-CoV-2 and the pangolin CoV MP789, both sharing high sequence identity, along with the Bat CoVs LYRa11, WIV1 and Rs7327, which are more related to the SARS-CoV S protein (Fig. 3A) . On the other hand, the deamidation hotspot at position 481 is fully exposed in the RBD, positioned in the external wall of the RBR (Fig. 3B and C) and

does not directly participate in hACE2 binding. The deamidation hotspot 481 is also present in the closely related SARS-CoV-2, the Bat CoV RaTG13 and the two pangolin viruses CoVsGX-P5E and MP789 (Fig.   3A ).

The Bat CoVRs/YN2018A, which was included as a representative Sarbecovirus that lacks a RBR and does not bind hACE2, shows a predicted deamidation hotspot at position 487 (SI Appendix, Table S7 ) that cannot be aligned with the RBR bearing sequences (Fig. 3A) . The superposition of the SARS-CoV-2 RBD (pdb: 6M0J) and RBD from the Bat CoVRs/YN2018A obtained by homology modeling shows that the 481 and 487 hotspots are located in a similar position in the three-dimensional structure of the domain, suggesting that topology drives conservation of deamidation-prone asparagine residues (Fig. 3C) .

Deamidation of the S protein RBM takes place in the context of the SARS-CoV2 life cycle. We have contextualized the results obtained for Asn 481 and 501 by numerical simulation and graphical representation of the SARS-CoV2 virion. We used the Gillespie algorithm as implemented in COPASI (38) to simulate the stepwise irreversible transition of the 33 S protein trimers in a virion (39, 40) from the intact state to the fully deamidated state. Our model considers deamidation of only Asn 481 and 501, with independent experimental half-time at 37 ºC (Fig. 2) . The six deamidation sites in each S trimer lead to 2 6 possible deamidation states, which can be grouped into 20 species using symmetry considerations (SI Appendix, Fig. S3 ).

We performed 1000 stochastic simulations and reported the average and standard deviation of the results for each time point. Supplementary Fig. S4 shows the full results, while Supplementary Table 8 heavily affected by temperature and the intact S trimer t1/2 decay at 4 ºC is delayed to 110 days. Due to the multimeric nature of both the virion and the Spike protein, trimers with at least one deamidated hotspot increase early:after only one day, close to 4 S trimers would host a deamidated hotspot (mainly at Asn 481). After two days, close to 7 of the S trimers have deamidated at one site and close to an additional trimer has deamidated at two sites (SI Appendix, Table S8 ). Fig. 4B shows a visualization of the deamidation state of the SARS-CoV2 virion after 48 h (41). Only 17 S protein trimers are shown for clarity (41) , while the population of each species is proportional to that in SI Appendix, Table S8 .

The intact S trimers are around four after two weeks, while the full deamidation of the virion takes about 1000 days. This timescale is far slower than that of SARS CoV-2 inactivation in solution at 37 ºC, (42) , indicating that deamidation of RBM hotspots is not the sole molecular mechanism for viral inactivation.

This does not necessarily imply that SARS CoV-2 receptor binding is not modulated by deamidation, since a quantitative model for the effect of deamidation should include other factors such as deamidation of other sites in S, changes in RBM receptor affinity upon deamidation (Fig. 2) , avidity effects arising from the trimeric nature of the S protein, modulation of the open/closed equilibrium of the trimer by deamidation, the presence of more than 30 S trimers in each virion and the presence of multiple receptor molecules on the surface of the target cell. Additionally, we would like to note that deamidation may have significantly altered the surface properties of the virion only two days after synthesis and this may have consequences for immune evasion.

Deamidation can critically affect protein performance and is of major importance in biologics and vaccine development, particularly when deamidating-prone asparagine residues are located in protein-protein binding interfaces or in antigenically relevant epitopes (43) (44) (45) .

The deamidation profile of the S proteins from a selected group of Betacoronaviruses shows a discrete number of predicted deamidation hotspots that are not randomly distributed over the entire length of the S ectodomain. Instead, deamidation hotspots are observed either fully conserved or clustered within the RBM. The RBM cluster is of particular importance due to its role in hACE2 binding and includes hotspots at positions 481, 493 and 501 that are partially conserved among Sarbecovirus. Deamidation hotspots in the RBM cluster that are critical for hACE2 binding are frequently mutated in naturally emerging variants of SARS-CoV or the SARS-CoV-2, suggesting that they are particularly sensitive to selective pressure.

We experimentally observed that Asn 481 deamidates with a half-time of 16.5 days at 37˚C, a reaction that is highly temperature sensitive. An aged RBD sample, in which 65% of the 481 hotspot is observed in its deamidated form showed a 3.5-fold increase of the Kd for hACE2. However, the idea that deamidated residues are always detrimental for protein fitness collides with the observation that hotspot 481 is The deamidation hotspot at position 501 is in direct contact with the K353 residue in hACE2 (32) and different observations reveal its critical role in determining hACE2 affinity (46, 47) .The N501Y mutation that eliminates this deamidation hotspot is a hallmark of the emerging SARS-CoV-2 variants, such as the In our experimental setup we did not find any significant deamidation of Asn 501 in the SARS CoV-2 RBD construct. However, experimental evidence about deamidation of Asn 501 was reported previously for this protein (26) . Such discrepancies may arise in the use of different experimental conditions that may affect protein conformation and hence, the deamidation rate. Alternatively, deamidation at these sites might not occur in the native, well folded S protein, but might be rapidly triggered when the protein is proteolyzed during the antigenic presentation process, interfering with the host-immune response (45) . Our integral kinetic model for the virion deamidation illustrates how the multimeric nature of S protein and the virion affect the accumulation of deamidate species. The model considers only two hotspots with their respective observed experimental deamidation half-times. The model shows that deamidation would not be the only molecular mechanism that reduces virus infectivity. The full deamidation time of the virion in which all of its 33 S trimers of the external surface are deamidated at both hotspots is close to 1000 days, far slower than the time required to eradicate the virus infectivity by the sole incubation at 37˚C, which has been reported to be of a couple of days (42) . However partial deamidated species are readily accumulated within hours and after a day close to four trimers would host a deamidated protomer, mainly at Asn 481.

This supports the possibility that deamidated hotspot can affect Spike fitness. For example, deamidation supports the co-existence of chemically diverse RBM populations (i.e, with different primary sequence) within the virion, some of which may enable the virus to evade antibody recognition. In this line, the E484K mutation (that coexist with the N501Y in the Brazilian SARS CoV-2 P.1 lineage (48), located nearby to Asn 481 in the RBR, has been associated with antibody escape suggesting that mutations or modifications in the RBR region may be related to an immune evasion viral mechanism (49) (50) (51) .This let us speculate that conservation of deamidation hotspots in topologically equivalent positions pursues some functionality.

One might speculate that the more infective emerging variants improve their fitness by eliminating a deamidation-prone residue that, when deamidated, decrease the affinity for the receptor. On the other hand, deamidation hotspot at position 493 (Asn 479 in the SARS-CoV sequence), which is in direct contact to K31 in hACE2 (9, 32) , was only observed in SARS-CoV GZ0402, which has been isolated from a handful of individuals. Unlike, the most common SARS-CoV Urbani that possesses the dipeptide Asn-Asp at positions 479 and 480, which is not expected to be a deamidation hotspot, the SARS-CoV GZ0402 bears a Asn-Gly hotspot. Interestingly, a deamidation hotspot is generated in antibody escape mutants of SARS-CoV recovered under the selective pressure of the R80 neutralizing monoclonal antibody. The escape variants bear a D480G mutation that transforms a ND slow-deamidating Asn into a NG deamidation hotspot (52, 53) . In this regard, the deamidation profile may be useful for leveraging worldwide sequence datasets aiming at prioritizing the early identification of escape variants of CoVs.

Deamidation estimation using NGOME-LITE.

The protein sequences of five S proteins were selected to investigate the presence of deamidation hotspots (SARS-2, SARS-URBANI, SARS-GZ0402, Bat-RaTG13 and Bat-WIV1). We used NGOME-LITE (23) to predict deamidation propensity of Asn residues by using predicted t1/2 values. To compare sites of deamidation, the protein sequences were aligned using Clustal Omega (54) with default parameters. The residue numbering of SARS-CoV-2 S were used as reference.

The SARS CoV-2 RBD (GenBank: MN908947) was expressed and purified as reported in (30) . The RBD (residues 319 -566) was expressed by transient transfection in HEK293-F containing a secretion signal, a C-terminal Sortase motif and a non-cleavable Histidine tag. Transfected cells were incubated at 37 ° with agitation at 220 rpm and 8 % CO2 atmosphere. Media containing secreted protein was harvested 4 days post transfection. During this "production" time, deamidation should occur adding a cumulative amount of deamidated species at zero time. All steps of the purification were performed at 4 °C, during which deamidation rate is expected to be considerably reduced. For RBD purification, an initial IMAC step was performed on a His-Trap column (all columns by GE Healthcare) using buffers A (50 mM Tris-HCl, 0.5 M NaCl, 10 mM imidazole, pH 7.4) and B (same as buffer A but with 500 mM imidazole). An additional gel filtration step was performed on a Superdex 200 Increase 10/300 GL column equilibrated with 50 mM Tris-HCl, 150 mM NaCl, pH 7.4. The protein was concentrated to 10 mg/ml and flash frozen in liquid nitrogen and stored at −80 °C until further use.

A gene encoding hACE2 residues 1-615 followed by a C-terminal HRV 3C cleavage site and a TwinStrepII-tag was cloned in the pXLG expression vector. HEK293F cells transfected with this construct were grown in a TubeSpin® bioreactor in FreeStyle293 medium for 72h at 37°C with 8% CO2 and agitation at 180 rpm. The secreted hACE2 protein was purified from the cell culture medium using a 1 ml StrepTactin Superflow high capacity cartridge (IBA). After elution, the C-terminal TwinStrepII-tag was removed by cleavage with His6-tagged HRV 3C protease, followed by an IMAC step to remove the HRV 3C protease from the sample. Finally, the untagged hACE2 protein was injected into a Superdex200

Increase 10/300 GL size exclusion chromatography column (Cytiva) equilibrated in 50 mM Hepes pH 7.2, 150 mM NaCl and 10% glycerol. The protein was concentrated to 1.9 mg/ml, flash-frozen in liquid nitrogen and stored at -80°C.

hACE2 was chemically biotinylated using the EZ-Link NHS-PEG4-Biotin kit (Sigma). The sample was first desalted using a PD-10 gravity flow column (GE Healthcare) in 20 mM sodium phosphate buffer nd 150 mM NaCl, pH 7.4. Subsequently, the sample was chemically biotinylated for 2 h on ice, using a 20-fold molar excess of biotin over the target protein. Excess biotin was removed by running the sample through a Size Exclusion Chromatography column (Superdex 200 Increase 10/300 GL). The fractions containing hACE2 were collected and concentrated to approximately 1 mg/ml. 5% v/v glycerol was added before flash-freezing, and the samples were stored at -80 °C until further use.

Purified RBD sample were diluted to 1 mg/ml in buffer 50 mM Tris-HCl, 150 mM NaCl and 5% v/v glycerol, pH 7.4, filtered with a pore size of 0.22 µm for minimizing bacterial growth and added in sealed SafeSel vials that were further sealed with parafilm. Samples were incubated at 4°C and 37°C. Independent aliquots were taken at different times for deamidation quantitation.

Digestion was done at pH 5.7 to avoid extensive deamidation during sample processing. Protein samples were diluted 1:10 in 100mM ammonium acetate (Fluka), pH 5.7, containing 10% v/v acetonitrile (Fisher Scientific). Disulfide bonds were reduced with 1 mM DTT (Sigma) and 1 mM TCEP (Invitrogen) for 30 min at 37°C. Proteins were first digested with trypsin (Promega) for 1 h at 37°C (sample to enzyme ratio 1:20) and then with GluC (Promega) (1:40 ratio) for additional 3h at 37°C. The peptides were cleaned up using an OASIS HLB µElution Plate (Waters).

An UltiMate 3000 RSLC nano LC system (Dionex) fitted with a trapping cartridge (µ-Precolumn C18 PepMap 100, 5µm, 300 µm i.d. 

Raw data were searched against the Uniprot human (75,069 entries, June 2020) and the SARS-CoV-2 (14 entries, June 2020) reference proteome using MaxQuant 1.6.14.0 (31). The default parameters were used with the following adjustments: Deamidation of Asn and Gln, ammonia-loss at Asn were set as variable modifications, as well as oxidation of Met and acetylation of protein N-termini. The minimum peptide length was adjusted to 6 and the maximum peptide mass to 8000 Da. For digestion Trypsin/P and GluC were enabled and the missed cleavages were set to 3. Label-free quantification was enabled, with Fast LFQ disabled. IBAQ calculations were enabled. The minimum number of unique peptides was set to 1.The Maxquant output msms.txt was imported into Skyline 20.2.0.286 (55) . Precursor ion chromatograms were extracted and the values of the peak areas were exported for further calculations. MS1 spectra were inspected manually using Thermo Xcalibur Qual browser 4.2.28.14.

The binding of RBD (0 days after purification or 20 days after deamidation at 37°C) to biotinylated hACE2 was measured by biolayer interferometry (BLI) using the Octet RED96 system (FortéBio). Concentration-dependent kinetic assays were performed by loading biotinylated hACE2 on streptavidin biosensors (FortéBio), pre-equilibrated in assay buffer (PBS buffer supplemented with 0.1% (w/v) BSA and 0.02%

(v/v) Tween-20) for 15 min. Prior to the association, a baseline step of 300 s was performed.

Subsequently, sensors were dipped in a different well containing 200, 100, 50, 25 and 12.5 nM of RBD for 600 s followed by 900 s of dissociation time in the same buffer. All experiments were carried out at 30 °C.

Data were reference-subtracted only for the association and aligned with each other with an in-house python script, using a 1:1 binding model. All figures were prepared using R and ggplot2. Two independent experiment were done. Deamidation hotspots on the SARS-CoV-2 S protein are shown in red. Protomers are colored in different gray tones. The PDB 6zgg (24) was used for this analysis, which contains a furin-cleaved SARS-CoV-2 S trimer, with two RBDs in the down conformation (chains A and C) and one in the up conformation (chain B). D) Top view of C), showing exposed deamidation hotspots at the RBD (Asn 481 and 501). E) Zoom of the exposed Asn 544 residues. 

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Deamidation and isoaspartate formation in proteins:unwanted alterations or surreptitious signals?

Deamidation of Amino Acids on the Surface of Adeno-Associated Virus Capsids Leads to Charge Heterogeneity and Altered Vector Function

Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation

Machine Learning Enables Accurate Prediction of Asparagine Deamidation Probability and Rate

Quantifying the Risks of Asparagine Deamidation and Aspartate Isomerization in Biopharmaceuticals by Computing Reaction Free-Energy Surfaces

Prediction of protein deamidation rates from primary and threedimensional structure

Molecular clocks. Proc. Natl. Acad. Sci

Proteome-wide prediction of spontaneous protein deamidation highlights differences between taxa

SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects

SARS-like WIV1-CoV poised for human emergence

Virus-Receptor Interactions of Glycosylated SARS-CoV-2 Spike and Human ACE2 Receptor

Protein asparagine deamidation prediction based on structures with machine learning methods

Structure Based Prediction of Asparagine Deamidation Propensity in Monoclonal Antibodies

Beyond Shielding: The Roles of Glycans in SARS-CoV-2 Spike Protein. bioRxiv

Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2

MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

Structure of SARS Coronavirus Spike Receptor-Binding Domain Complexed with Receptor

The evolutionary history of ACE2 usage within the coronavirus subgenus Sarbecovirus. bioRxiv

Virus taxonomy: the database of the International Committee on Taxonomy of Viruses (ICTV)

Structural basis of receptor recognition by SARS-CoV-2

SARS-CoV-2 Variants.WHO -Dis. Outbreak News

COPASI-a COmplex PAthway SImulator

Structures and distributions of SARS-CoV-2 spike proteins on intact virions

SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography. bioRxiv

Integrative illustration for coronavirus outreach

Stability of SARS-CoV-2 in different environmental conditions

Altered immunogenicity of isoaspartate containing proteins

Deamidation of Asparagine in a Major Histocompatibility Complex-Bound Peptide Affects T Cell Recognition but Does Not Explain Type B Reactivity

Asparagine Deamidation Perturbs Antigen Presentation on Class II Major Histocompatibility Complex Molecules *

Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies

Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding

Emergence of SARS-CoV-2 B.1.1.7 Lineage -United States

Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and N501Y variants by BNT162b2 vaccine-elicited sera

Molecular determinants and mechanism for antibody cocktail preventing SARS-CoV-2 escape

Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies

Broadening of Neutralization Activity to Directly Block a Dominant Antibody-Driven SARS-Coronavirus Evolution Pathway

Effects of Human Anti-Spike Protein Receptor Binding Domain Antibodies on Severe Acute Respiratory Syndrome Coronavirus Neutralization Escape and Fitness

Clustal Omega for making accurate alignments of many protein sequences

Platform-independent and Label-free Quantitation of Proteomic Data Using MS1 Extracted Ion Chromatograms in Skyline: APPLICATION TO PROTEIN ACETYLATION AND PHOSPHORYLATION *

Experimental deamidation kinetic and affinity to hACE2 of an aged RBD sample. A) Left

Time-decay of asparagine-containing peptides for the 481, 501 and 544 hotspots at 4 ˚C. The lines are fit to an exponential function with initial amplitude of 100% and endpoint of 0%. Right: Time-decay of asparagine-containing peptides for the 481, 501 and 544 hotspot at 37 C. The lines are fits to anC. The exponential decay. B) Left: BLI response curves of a fresh RBD sample to an immobilized hACE2

Acknowledgments and funding sources. under Marie Curie COFUND actions. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.